This invention relates to a regulatable and replicable expression and secretion vector useful for B. subtilis and other gram-positive bacteria.
Several expression and secretion vectors for heterologous protein production in Bacillus subtilis have been reported. These include .alpha.-amylase [Palva et al., Proc. Natl. Acad. Sci. USA 79:5582-5586 (1982); Palva et al., Gene, 22:229-235 (1983), and GB 2,091,268B] and protease gene-based vectors [Vasantha et al., J. Bacteriol., 165:837-842, (1986), Honjo et al., J. Biotech 4:63-71 (1986), and Nagarajan et al., U.S. Pat. No. 4,801,537]. A disadvantage of these vectors is that their expression is not regulated, and the heterologous protein is produced at all times. A B. subtilis secretion vector based on the levansucrase gene from B. subtilis (sacB[Bsu]) has also been reported by Joyet et al., in Bacillus: Molecular Genetics and Biotechnology Applications, eds. Ganesan & Hoch (Academic Press, 1986), 479-493. Edelman et al., FEMS Microbiology Letters 52:117-120 (1988) discloses such a vector which is replicable. However, the levansucrase gene on a multicopy plasmid vector is homologous to the chromosomal sacB[Bsu] gene of B. subtilis bacteria; therefore, extensive recombination may occur and result in plasmid instability. Thus there is a need for a stable, regulatable expression vector for cloning heterologous genes into B. subtilis and other gram positive bacteria which can overcome these disadvantages.
The Bacillus subtilis levansucrase expression system has several advantages as the basis for designing a regulatable expression system. It is regulatable by sucrose, which is inexpensive and easily purified from the protein product. However, a major obstacle which exists for genetic engineering of the levansucrase expression system is its complexity. There are at least two other genes present in the levansucrase regulon, sacS and sacU (Lepesant et al., in Microbiology, American Society of Microbiology [1976]; Debarbouille et al., FEMS Microbiol. Lett. 41, 137-140 [1987]), which are involved with the control of expression of the gene. The structural gene is transcriptionally regulated in a way or ways which are not well understood, but which may be related to the formation of particular structures in the regulatory region of the gene between the transcription initiation sequence and the translation start sequence. These structures may be destabilized by the very precise and delicate interactions between the products of the sacS and sacU genes in the presence of sucrose, thus causing antitermination and readthrough into the structural gene (Shimotsu et al., J. Bacteriol. 168:380-388 [1986]). Thus any attempts at genetically engineering the levansucrase expression system are complicated by the need to take into account compatibility with this complex regulon.
The mechanism of sucrose regulation of sacB[Bsu] has been studied by Shimotsu et al., suora. The DNA sequence of the regulatory region between the promoter and start site of translation consists of inverted repeats. Thus, the RNA structure has the potential to form a long stem and a short loop structure. Shimotsu et al. also determined the DNA sequence of two B. subtilis mutants that were no longer regulated by sucrose (levansucrase was expressed independently of sucrose). The mutations in both cases were found to be single-base changes in the regulatory region (see FIG. 4). Thus there was some indication that the regulation of levansucrase is very tightly controlled by the specific DNA sequence of this regulatory region in B. subtilis.
It is also known that at least one other species of Bacillus, e.g. Bacillus amyloliquefaciens, made a levansucrase (Mantsala, P. and M. Puntab, FEMS Microbiol. Lett. 13, 395-399 [1982]), but it was not known whether it was regulated in B. amyloliquefaciens, or, if so, whether the expression elements of the B. amyloliquefaciens gene would be compatible with the other genes of the B. subtilis sucrose regulon.